1. Field of the Invention
This invention relates to a fluorescence sensor for detecting a specific substance, which is contained in a sample, by use of a fluorometric analysis technique.
2. Description of the Related Art
Heretofore, in fields of biological analyses, and the like, a fluorometric analysis technique has been used widely as an analysis technique, which has a high sensitivity and is easy to perform. The fluorometric analysis technique is the technique, wherein exciting light having a specific wavelength is irradiated to a sample expected to contain a substance to be detected, which substance is capable of producing fluorescence by being excited by the exciting light having the specific wavelength, wherein the fluorescence having thus been produced by the substance to be detected is detected, and wherein the presence of the substance to be detected is thereby confirmed. In cases where the substance to be detected is not a fluorescent substance, a technique has heretofore been conducted widely, wherein a specific binding substance, which has been labeled with a fluorescent substance and is capable of undergoing the specific binding with the substance to be detected, is brought into contact with the sample, wherein the fluorescence is detected in the same manner as that described above, and wherein the occurrence of the specific binding, i.e. the presence of the substance to be detected, is thereby confirmed.
FIG. 2 is a schematic side view showing an example of a conventional fluorescence sensor for carrying out a fluorometric analysis technique utilizing a labeled specific binding substance. By way of example, the fluorescence sensor illustrated in FIG. 2 is utilized for detecting an antigen 2, which is contained in a sample 1. The fluorescence sensor illustrated in FIG. 2 comprises a base plate 3, on which a primary antibody 4 capable of undergoing the specific binding with the antigen 2 has been coated. The fluorescence sensor also comprises a sample support section 5, which is formed on the base plate 3. The sample 1 is caused to flow within the sample support section 5. A secondary antibody 6, which has been labeled with a fluorescent substance 10 and is capable of undergoing the specific binding with the antigen 2, is then caused to flow within the sample support section 5. Thereafter, exciting light 8 is irradiated from an exciting light source 7 toward a surface area of the base plate 3. Also, an operation for detecting the fluorescence is performed by a photodetector 9. In cases where the predetermined fluorescence is detected by the photodetector 9, the specific binding of the secondary antibody 6 and the antigen 2 with each other, i.e. the presence of the antigen 2 in the sample, is capable of being confirmed.
In the example described above, the substance whose presence is actually confirmed with the fluorescence detecting operation is the secondary antibody 6. If the secondary antibody 6 does not undergo the specific binding with the antigen 2, the secondary antibody 6 will be carried away and will not be present on the base plate 3. Therefore, in cases where the presence of the secondary antibody 6 on the base plate 3 is detected, the presence of the antigen 2, which is the substance to be detected, is capable of being confirmed indirectly.
The confirmation of the presence of the secondary antibody 6 on the base plate 3 is also capable of being made with a technique wherein, instead of the photodetector 9 being used, the fluorescence is detected with visual observation made by persons. For example, in cases where a simple type fluorescence sensor, such as a fluorescence sensor for domestic use, is to be formed, the constitution in which the photodetector is not provided is capable of being employed appropriately, such that the cost may be kept low.
Particularly, with the rapid advances made in enhancement of performance of photodetectors, such as the advances made in cooled CCD image sensors, in recent years, the fluorometric analysis technique described above has become the means essential for biological studies. The fluorometric analysis technique has also been used widely in fields other than the biological studies. In particular, with respect to the visible region, as in the cases of FITC (fluorescence wavelength: 525 nm, quantum yield: 0.6), Cy5 (fluorescence wavelength: 680 nm, quantum yield: 0.3), and the like, fluorescent dyes having high quantum yields exceeding 0.2, which serves as a criterion for use in practice, have been developed. It is thus expected that the fields of the application of the fluorometric analysis technique will become wide even further.
However, with the conventional fluorescence sensor as illustrated in FIG. 2, the problems are encountered in that noise is caused to occur by the reflected/scattered exciting light at an interface between the base plate 3 and the sample 1 and the light scattered by impurities/suspended materials M, and the like, other than the substance to be detected. Therefore, with the conventional fluorescence sensor, even though the performance of the photodetectors is enhanced, it is not always possible to enhance the signal-to-noise ratio in the fluorescence detecting operation.
As a technique for solving the problems described above, a fluorometric analysis technique utilizing an evanescent wave has heretofore been proposed. FIG. 3 is a schematic side view showing an example of a conventional fluorescence sensor for carrying out a fluorometric analysis technique utilizing an evanescent wave. In FIG. 3 (and in FIG. 1, which will be described later), similar elements are numbered with the same reference numerals with respect to FIG. 2. Accordingly, the explanation of the similar elements will hereinbelow be omitted.
In the fluorescence sensor illustrated in FIG. 3, in lieu of the base plate 3 described above, a prism (a dielectric material block) 13 is utilized. A metal film 20 has been formed on a surface of the prism 13. Also, the exciting light 8 having been produced by the exciting light source 7 is irradiated through the prism 13 under the conditions such that the exciting light 8 may be totally reflected from the interface between the prism 13 and the metal film 20. With the constitution of the fluorescence sensor illustrated in FIG. 3, at the time at which the exciting light 8 is totally reflected from the interface described above, an evanescent wave 11 oozes out to the region in the vicinity of the interface described above, and the secondary antibody 6 is excited by the evanescent wave 11. Also, the fluorescence detecting operation is performed by the photodetector 9 located on the side of the sample 1, which side is opposite to the side of the prism 13. (In the cases of FIG. 3, the photodetector 9 is located on the upper side.)
With the fluorescence sensor illustrated in FIG. 3, the exciting light 8 is totally reflected from the aforesaid interface downwardly in FIG. 3. Therefore, in cases where the fluorescence detecting operation is performed from above, the problems do not occur in that an exciting light detection component constitutes the background with respect to a fluorescence detection signal. Also, the evanescent wave 11 is capable of reaching only a region of several hundreds of nanometers from the aforesaid interface. Therefore, the scattering from the impurities/suspended materials M contained in the sample 1 is capable of being suppressed. Accordingly, the evanescent fluorometric analysis technique described above has attracted particular attention for serving as a technique, which is capable of markedly suppressing (light) noise than with the conventional fluorometric analysis techniques, and with which the substance to be detected is capable of being fluorometrically analyzed in units of one molecule.
The fluorescence sensor illustrated in FIG. 3 is the surface plasmon enhanced fluorescence sensor, which has the sensitivity having been enhanced markedly among the fluorescence sensors utilizing the evanescent fluorometric analysis technique. With the surface plasmon enhanced fluorescence sensor, wherein the metal film 20 is formed, at the time at which the exciting light 8 is irradiated through the prism 13, the surface plasmon arises in the metal film 20, and the fluorescence is amplified by the electric field amplifying effect of the surface plasmon. A certain simulation has revealed that the fluorescence intensity in the cases described above is amplified by a factor of approximately 1,000.
The surface plasmon enhanced fluorescence sensor of the type described above is described in, for example, Japanese Patent No. 3562912. Also, as described in, for example, “1. Total Internal Reflection Fluoresence Microscopy that Enables Observation of Surfaces Only with High Image Quality”, M. Tokunaga, Understanding with bio imaging, pp. 104-113, Yodosha, there has been known a fluorescence sensor, in which the fluorescence detecting operation is performed by use of the evanescent fluorometric analysis technique without the surface plasmon enhancement being utilized particularly. In such cases, the metal film 20 illustrated in FIG. 3 is omitted, such that the sample 1 may be in direct contact with the prism 13, and the fluorescent substance, such as the secondary antibody 6, is excited by the evanescent wave 11, which oozes out from the interface between the sample 1 and the prism 13.
In the cases of the surface plasmon enhanced fluorescence sensor, as described in, for example, “Surface Plasmon Field-Enhanced Fluorescence Spectroscopy Studies of the Interaction between an Antibody and Its Surface-Coupled Antigen”, F. Yu et al., Analytical Chemistry, Vol. 75, pp. 2610-2617, 2003, the problems occur in that, if the fluorescent substance contained in the sample and the metal film are markedly close to each other, energy having been excited in the fluorescent substance will undergo transition to the metal film before causing the fluorescent substance to produce the fluorescence, and a phenomenon of fluorescence production failure (i.e., the so-called metal quenching) will thus arise. As described in “Optical limiting effect in a two-photon absorption dye doped solid matrix”, G. S. He et al., Applied Physics Letters, Vol. 67, Issue 23, pp. 2433-2435, 1995, in order to cope with the metal quenching described above, a technique is proposed, wherein a self-organizing film (SAM) is formed on the metal film, and wherein the fluorescent substance contained in the sample and the metal film are spaced away from each other by a distance equal to at least the thickness of the SAM. In FIG. 3, the SAM is represented by the reference numeral 21.
With the fluorescence sensors as described above, wherein the difference (i.e., the so-called Stokes' shift) between the excitation wavelength for the fluorescent substance, which is currently utilized as the label, and the fluorescence wavelength is comparatively small, the problems are encountered in that the exciting light having been scattered by impurities contained in the prism is detected by the photodetector for the fluorescence detecting operation, and in that the signal-to-noise ratio of the measurement signal is thus not capable of being kept high. For example, in the cases of Cy5 described above, the fluorescence wavelength is 680 nm with respect to the excitation wavelength falling within the range of 635 nm to 645 nm, and the Stokes' shift is thus equal to at most approximately 40 nm. Therefore, ordinarily, at the time of the fluorescence detecting operation, a wavelength separation filter referred to as the sharp cut filter, such as a band pass filter, is located at a position just before the photodetector.
However, the wavelength separation capability of the aforesaid type of the filter is not sufficient for coping with the Stokes' shift as described above. Therefore, light noise often remains mixed in the measurement signal. Also, with the aforesaid type of the filter, which ordinarily has a markedly low transmittance, the problems occur in that the quantity of the fluorescence capable of being detected becomes small, and in that the signal-to-noise ratio of the measurement signal is thus caused to become low. Further, with the aforesaid type of the filter, the cost of which is high, the problems are encountered in that the cost of the fluorescence sensor is not capable of being kept low.
With the fluorescence sensor, in which the prism (the dielectric material block) is utilized and in which the fluorescent substance is excited by the evanescent wave, the problems described above are encountered. With a constitution, in which the prism is not utilized, in cases where the exciting light is detected by the photodetector due to certain reasons or in cases where the exciting light impinges upon the eyes of the sensor operator for visually detecting the fluorescence, the accuracy of the fluorescence detecting operation is affected adversely.